The overall goal of this research is to develop a mechanistic understanding of the vacuolar biogenesis pathway in the yeast Saccharomyces cerevisiae. Genetic analysis has lead to the identification of a very large number of components required along the vacuolar assembly pathway in yeast, and the major focus of this proposal is a functional characterization of Vps-dependent membrane transport to the vacuole. The Vps 1 protein has been found to be a dynamin-like GTPase associated with the Golgi compartment, and cells lacking Vps1p route membrane traffic to the vacuole via the plasma membrane. Genetic and biochemical approaches will be pursued to identify proteins that interact with and regulate Vps1p function. Proteins responsible for regulating the GTPase cycle of Vps1p will be identified and characterized to help reveal the mechanistic details of this fascinating dynaMin-like, Golgi-associated GTPase. Vps45p is a member of the Sec 1p family of proteins and found to be required for the consumption of post-Golgi, vacuole-targeted vesicles, and as such is predicted to be part of a SNARE complex. Combining genetic and biochemical approaches we will identify the other components of the Vps45p-containing SNARE complex, determine their inter-relationship and investigate the precise execution point for this complex in the vacuolar biogenesis pathway. Vps27p and other class E Vps proteins have been found to be required for membrane traffic out of the yeast prevacuole/late-endosome. Using density gradient centrifugation and immunoisolation approaches we will isolate the membrane transport vesicles that rapidly and reversibly accumulate in vps45-ts mutant cells and the prevacuole/late-endosomal compartment that again rapidly and reversibly accumulates in vps27-ts cells. Biochemical approaches will be employed to investigate the content of these organelles and to identify the various Vps proteins that are predicted to be associated with these membranes. Another major focus of this proposal is to investigate the roles of the individual members of the family of yeast vacuolar protein sorting receptors. The CPY sorting receptor, Pep 1p, has been identified and two other highly related receptor-like proteins were also identified. These sorting receptors will be characterized for their ability to sort the various vacuolar hydrolases present in yeast. The ability of these receptor proteins to cycle between the Golgi apparatus and the prevacuole/late-endosome will be investigated in the various vps mutants in our collection. Efforts to identify proteins that regulate the targeting and function of the CPY sorting receptor lead to the identification of the PTI1 gene, which encodes a syntaxin-like membrane protein required not only for vacuolar protein sorting but also for haploid yeast cell growth. pti1-ts yeast mutants will be studied to reveal the fate of the vacuolar protein sorting receptors and to shed light on the unexpected haploid lethal phenotype associated with disruption of PTI1.